The immunoregulatory functions of macrophages in the afferent phase of cell mediated immune (CMI) responses has received little attention and the mechanism by which these cells function in the induction of cell mediated cytotoxic responses is unknown. While it is clear that this cell is important in regulating the cellular events of antibody formation, the role of the macrophage in the induction thymus derived (T) cells is less clear. The macrophage has a critical, although yet undefined, role in the induction of classical skin test cell mediated immunity (CMI) in vivo and various models of CMI in vitro. The mitogenic and allogeneic activation of T cells has been shown to be dependent on macrophages but the exact mechanism by which these cells function in the above phenomena is unknown. We propose to determine the mechanism by which macrophages regulate the afferent phase of the generation of antitumor cell mediated cytotoxicity (CMC). Preliminary data suggest that macrophages act as: 1) stimulator cells, 2) accessory cells, 3) cells capable of presenting bound antigen to responder cells, and 4) cells possibly secreting soluble immunoregulatory products. We have developed two in vitro model systems to explore the mechanisms of immunoregulation by macrophages in the afferent phase of the generation of CMC. Using murine spleen lymphocytes, we have developed one system to generate and measure CMC induced by allogeneic combinations of lymphocytes. In the other, we generate syngeneic, tumor specific CMC when C57BL6 spleen cells are stimulated by the syngeneic EL4 lymphoma cell. We have shown, on a preliminary basis, that macrophages are required for the generation of CMC in both systems. In addition, the presentation of macrophage bound alloantigen and syngeneic tumor specific antigen is quantitatively more efficient in inducing CMC than equal numbers of free allogeneic lymphocytes or syngeneic tumor cells.